Major Caffeine Metabolites Research Articles

The Role of Nitric Oxide in the Proconvulsant Effect of Paraxanthine During the Intravenous Pentylenetetrazole Seizure Threshold Test in Mice

Background: Paraxanthine is the major metabolite of caffeine in humans. There is no definitive proof that paraxanthine affects seizures. Nitric oxide (NO) contributes to the central effects of paraxanthine. Objectives: In this study, we examined the effect of acute paraxanthine administration on the pentylenetetrazole (PTZ)-induced seizure threshold, focusing on the NO-cyclic guanosine monophosphate (cGMP) signaling pathway. Methods: Naval Medical Research Institute (NMRI) male mice (25 - 30 g) received paraxanthine (5, 10, 50, and 100 mg/kg) alone. L-arginine [a substrate for NO synthase (NOS)] (50 mg/kg) or L-NAME [a non-selective NOS inhibitor] (5 mg/kg) was administered alone or as pretreatment before the ineffective or effective doses of paraxanthine, respectively. The intravenous PTZ seizure threshold test was used to determine the thresholds for the onset of myoclonic twitch (MCT), generalized clonus (GNC), and forelimb tonus (FLT). Nitric oxide metabolites (NOx) were measured using the Griess method. Results: Paraxanthine administered at doses of 10 and 50 mg/kg significantly reduced the threshold for FLT (P < 0.05 for both). At a dose of 100 mg/kg, paraxanthine significantly reduced the thresholds for the onset of MCT (P < 0.001), GNC (P < 0.01), and FLT (P < 0.001). L-arginine (50 mg/kg), either alone or as pretreatment before paraxanthine (5 mg/kg), did not significantly change the seizure threshold. L-NAME (5 mg/kg) alone had no effect, but L-NAME pretreatment before paraxanthine (100 mg/kg) significantly increased the thresholds for the onset of MCT (P < 0.001), GNC (P < 0.001), and FLT (P < 0.001). Only paraxanthine at a dose of 100 mg/kg significantly increased NOx levels in brain tissues (P < 0.05). Pretreatment with L-arginine further increased the NOx level (P < 0.001), whereas pretreatment with L-NAME decreased it (P < 0.001) compared to the paraxanthine groups. Conclusions: Our results show that paraxanthine has a proconvulsant effect. The results of L-arginine or L-NAME pretreatment before paraxanthine support the possible interaction of the NO-cGMP pathway in the proconvulsant effect of paraxanthine.

Urinary caffeine and its metabolites in association with advanced liver fibrosis and liver steatosis: a nationwide cross-sectional study.

Aim: This study used urinary caffeine and its metabolites to evaluate their relationships with liver steatosis and advanced liver fibrosis. Methods: A total of 2068 adult participants with required data were filtered from the 2009-2014 National Health and Nutrition Examination Survey (NHANES) cycles. Non-invasive scores were applied to define liver steatosis and advanced liver fibrosis. Logistic regression models, weighted quantile sum (WQS) regression models, quantile-based g-computation (QG-Comp) models, and restricted cubic spline (RCS) regression models were used to assess the associations of urinary caffeine and its metabolites with liver steatosis and advanced liver fibrosis. A series of additional analyses were conducted to examine the subgroup-specific differences and test the robustness of the observed results. Results: The major caffeine metabolite mixture and most individual caffeine metabolites were found to be negatively associated with the risk of advanced liver fibrosis with subgroup-specific variations. Only 7-MX consistently showed a negative association with liver steatosis in all analyses, while no association was observed between the major caffeine metabolite mixture and liver steatosis. Conclusion: The major caffeine metabolite mixture and most individual urinary caffeine metabolites exhibited inverse associations with advanced liver fibrosis with subgroup differences. Further prospective and experimental studies are urgently needed to verify our results and further identify the possible mechanisms.

Exploring Partial Structural Disorder in Anhydrous Paraxanthine through Combined Experiment, Solid-State Computational Modelling, and Molecular Docking

Paraxanthine (PX), a major metabolite of caffeine, a protective agent against Alzheimer’s and Parkinson’s disease, and a promising drug for the treatment of post-COVID 2019 anosmia and ageusia, has been studied in the solid state and protein–ligand complex. Partial disorder in PX, caused by the methyl group at the N(7) position, has been modelled and discussed. The relationship between the unusual structural disorder and the propensity to form a specific system of non-covalent bonds was analyzed. Three 1H-14N NMR-NQR (nuclear magnetic resonance–nuclear quadrupole resonance) experimental techniques were used, namely multiple frequency sweeps, Larmor frequency scanning, and the two-frequency irradiation, followed by solid-state computational modelling (density functional theory, supplemented by quantum theory of atoms in molecules, 3D Hirshfeld surfaces, and reduced density gradient), and molecular docking approaches. New quantitative methods for estimating changes in the global pattern of interactions under the influence of rotation of the methyl group in N(7) based on the Pompeiu–Hausdorff and Bhattacharayya metrics and the Wasserstein distance have been proposed and applied. A spectrum consisting of 12 lines, indicating the presence of 4 chemically inequivalent nitrogen sites in the PX molecule, was recorded, and the lines’ assignment to particular sites was made. The influence of the methyl rotation on the eigenvalues and eigenvectors of the electric field gradient tensor, NQR parameters, and resonance line positions was modelled in the solid (GGA/RPBE, m-GGA/RSCAN) and cluster (Minnesota M062X hybrid). Three factors have been found to determine structural disorder in PX: larger crystal voids near the methyl at N(7) than at N(1) (opening the path for the disorder), hyperconjugation strongly affecting the density distribution in the five-membered ring, and the involvement of the methyl group at N(7) in many non-covalent bonds that intercept (capture) subsequent jumping protons. The Pompeiu–Hausdorff and Bhattacharayya metrics and the Wasserstein distance confirmed the changes in the distribution and strength of non-covalent interactions throughout the molecule as a result of methyl rotation. This effect is clearly visible regardless of the type of metric, and its order of magnitude is consistent with the modulation effect of the NQR spectra (experimental and calculated). Through molecular docking, it was discovered that the PX moiety in protein–ligand complexes adopt the same methyl group conformation at N(7) as in the solid state. It was found that the cooperation–competition between the C-H⋯O hydrogen bonds and C-H⋯H-C dispersion interactions is the crucial factor that impedes methyl rotation and induces structural disorder, as well as being an important factor in the formation of the protein–ligand complexes.

A purine derivative, paraxanthine, promotes cysteine uptake for glutathione synthesis

Purine derivatives such as caffeine and uric acid have neuroprotective activities and are negatively correlated with the incidence of both Alzheimer's disease and Parkinson's disease. We have reported that an increment of intracellular glutathione (GSH) via cysteine uptake in neuronal cells is one of the mechanisms by which caffeine and uric acid confer neuroprotection. Here, we investigated whether caffeine metabolites such as paraxanthine, theophylline, theobromine, 1,7-dimethyluric acid and monomethylxanthines would increase cysteine uptake in mouse hippocampal slices. The metabolites were administered to hippocampal slices for 30 min at doses of 0, 10, or 100 μM, and then cysteine was added for 30 min. Paraxanthine, a major metabolite of caffeine, increased cysteine content in the slices, whereas the other metabolites did not. In vitro treatment with paraxanthine promoted cysteine uptake and increased GSH in HEK293 cells. The paraxanthine-induced cysteine uptake was inhibited by an excitatory amino-acid carrier-1 (EAAC1) inhibitor, and H2O2-induced cell damage was prevented by the paraxanthine treatment of SH-SY5Y cells. These results suggest that paraxanthine, an active metabolite of caffeine, acts to increase intracellular GSH levels via EAAC1 leading to neuroprotection.

Review Article on Caffeine Activity

Caffeine (1,3,7-trimethylxanthine) is the most consumed psychoactive substance in the world, acting by means of antagonism to adenosine receptors, mainly A1 and A2A. Coffee is the main natural source of the alkaloid which is quite soluble and well extracted during the brew’s preparation. After consumption, caffeine is almost completely absorbed and extensively metabolized in the liver by phase I (cytochrome P450) enzymes, mainly CYP1A2, which appears to be polymorphically distributed in human populations. Paraxanthine is the major caffeine metabolite in plasma, while methylated xanthine and methyl uric acids are the main metabolites excreted in urine. In addition to stimulating the central nervous system, caffeine exerts positive effects in the body, often in association with other substances, contributing to prevention of several chronic diseases. The potential adverse effects of caffeine have also been extensively studied in animal species and in humans. Chemical structure of caffeine molecule.

Coffee effectively attenuates impaired attention in ADORA2A C/C-allele carriers during chronic sleep restriction

Many people consume coffee to attenuate increased sleepiness and impaired vigilance and attention due to insufficient sleep. We investigated in genetically caffeine sensitive men and women whether ‘real world’ coffee consumption during a simulated busy work week counteracts disabling consequences of chronically restricted sleep. We subjected homozygous C-allele carriers of ADORA2A (gene encoding adenosine A2A receptors) to five nights of only 5 h time-in-bed. We administered regular coffee (n = 12; 200 mg caffeine at breakfast and 100 mg caffeine after lunch) and decaffeinated coffee (n = 14) in double-blind fashion on all days following sleep restriction. At regular intervals four times each day, participants rated their sleepiness and performed the psychomotor vigilance test, the visual search task, and the visuo-spatial and letter n-back tasks. At bedtime, we quantified caffeine and the major caffeine metabolites paraxanthine, theobromine and theophylline in saliva. The two groups did not differ in age, body-mass-index, sex-ratio, chronotype and mood states. Subjective sleepiness increased in both groups across consecutive sleep restriction days and did not differ. By contrast, regular coffee counteracted the impact of repeated sleep loss on sustained and selective attention, as well as executive control when compared to decaffeinated coffee. The coffee also induced initial or transient benefits on different aspects of baseline performance during insufficient sleep. All differences between the groups disappeared after the recovery night and the cessation of coffee administration. The data suggest that ‘real world’ coffee consumption can efficiently attenuate sleep restriction-induced impairments in vigilance and attention in genetically caffeine sensitive individuals.German Clinical Trial Registry: # DRSK00014379.

Detection of caffeine intake by means of EC-SERS spectroscopy of human saliva.

This work presents the application of EC-SERS spectroscopy for the detection of caffeine consumption from human saliva. Caffeine and paraxanthine as the major metabolite of caffeine were tested. Model samples of saliva spiked with caffeine were investigated, and detection of caffeine in real-life saliva samples was tested in order to ensure the viability of the method for clinical applications. Two doses of caffeine (2 mg/kg and 3.5 mg/kg) were ingested by volunteers, and their saliva samples were taken at different time periods ranging from 1 h to 10 h after the consumption. Density functional theory calculations of caffeine and paraxanthine adsorbed on the silver surface were performed in order to better understand the adsorption of the investigated molecules and to make a correct assignment of the experimental spectral bands of the EC-SERS spectra. It was determined that a low dose caffeine consumption can be detected by the appearance of the SERS spectral marker band of caffeine and paraxanthine at 692 cm-1. The intensity of this band is mostly reasoned by the paraxanthine concentration since the intensity changes of the band over time correlates to the concentration changes of paraxanthine determined by the pharmacokinetic studies of paraxanthine and caffeine in the human saliva. It was found that the limit of detection paraxanthine in saliva by means of EC-SERS is as low as 15 μM and can be further improved.

Caffeine Consumption through Coffee: Content in the Beverage, Metabolism, Health Benefits and Risks

Caffeine (1,3,7-trimethylxanthine) is the most consumed psychoactive substance in the world, acting by means of antagonism to adenosine receptors, mainly A1 and A2A. Coffee is the main natural source of the alkaloid which is quite soluble and well extracted during the brew’s preparation. After consumption, caffeine is almost completely absorbed and extensively metabolized in the liver by phase I (cytochrome P450) enzymes, mainly CYP1A2, which appears to be polymorphically distributed in human populations. Paraxanthine is the major caffeine metabolite in plasma, while methylated xanthines and methyluric acids are the main metabolites excreted in urine. In addition to stimulating the central nervous system, caffeine exerts positive effects in the body, often in association with other substances, contributing to prevention of several chronic diseases. The potential adverse effects of caffeine have also been extensively studied in animal species and in humans. These aspects will be approached in the present review.

Caffeine and paraxanthine HPLC assay for CYP1A2 phenotype assessment using saliva and plasma

Caffeine has been extensively used as a probe to measure CYP1A2 activity in humans with caffeine clearance or the paraxanthine (major metabolite of caffeine) to caffeine concentration ratio being regarded as the preferred metric. A simple reverse-phased C(18) HPLC assay using ethyl acetate liquid-liquid extraction was developed to quantitate caffeine and paraxanthine concentrations in saliva and plasma. The mobile phase consisted of acetonitrile-acetic acid-H(2)O (100:1:899) and analytes were quantitated with UV detection at 280 nm. The extraction recovery for paraxanthine and caffeine was approximately 70% in both saliva and plasma. The assay was linear over the concentration ranges 0.05-2.50 and 0.05-5.00 µg/mL, for paraxanthine and caffeine, respectively, in saliva. In plasma the assay was linear over the ranges 0.025-2.50 and 0.025-5.00 µg/mL for paraxanthine and caffeine, respectively. Intra- and inter-assay precision and accuracy were less than 15%. Detection limits were 0.015 µg/mL for paraxanthine and caffeine in saliva, while it was 0.005 µg/mL for paraxanthine and caffeine in plasma. Utility was established in samples collected from two healthy volunteers who abstained from caffeine for 24 h and received a single 100 mg oral dose of caffeine. The assay developed is a robust, simple and precise technique to measure caffeine and paraxanthine in saliva and plasma of healthy volunteers after a single oral dose of caffeine.

Reduction in non-glomerular renal clearance of the caffeine metabolite 1-methylxanthine by probenecid

Urinary caffeine metabolic ratios used to quantify the activity of numerous drug-metabolizing enzymes are an established component of cocktail approaches for metabolic phenotyping. Because in vitro evidence suggests that 1-methylxanthine (1-MX), a major caffeine metabolite, is actively secreted into urine by organic anion transporters (hOATs), coadministration of renal hOAT inhibitors like probenecid may impair these procedures. In a randomized, placebo-controlled, double-blind crossover design, single oral doses of 300 mg caffeine with oral coadministration of placebo or 500 mg probenecid 3 times daily for 2 days were administered to 7 healthy men. The plasma and urine concentrations of caffeine and its major metabolites 1,7-dimethylxanthine (1,7-DMX) and 1-MX were determined by high-performance liquid chromatography. Coadministration of probenecid resulted in a 34% reduction of the renal clearance of 1-MX (mean +/- SD 190 +/- 42 versus 290 +/- 83 ml min(-1), 95% CI on difference 0.2, 200, p = 0.04) with a 41% reduction in its estimated non-glomerular clearance. The renal clearances of caffeine and 1,7-DMX and the area under the plasma concentration-time curves of all substances were not significantly changed. 1-MX undergoes renal tubular secretion which is substantially reduced by probenecid, possibly due to inhibition of renal hOATs. This inhibition may explain the influence of probenecid on urinary caffeine metabolic ratios and, thus, its impact on the assessment of enzyme activities. It also suggests that 1-MX might serve as a model substrate for the renal tubular transport of organic anions.

Caffeine metabolites are inhibitors of the nuclear enzyme poly(ADP-ribose)polymerase-1 at physiological concentrations

The activity of the nuclear enzyme poly(ADP-ribose)polymerase-1 (E.C.2.4.2.30), which is highly activated by DNA strand breaks, is associated with the pathophysiology of both acute as well as chronic inflammatory diseases. PARP-1 overactivation and the subsequent extensive turnover of its substrate NAD + put a large demand on mitochondrial ATP-production. Furthermore, due to its reported role in NF-κB and AP-1 mediated production of pro-inflammatory cytokines, PARP-1 is considered an interesting target in the treatment of these diseases. In this study the PARP-1 inhibiting capacity of caffeine and several metabolites as well as other (methyl)xanthines was tested using an ELISA-assay with purified human PARP-1. Caffeine itself showed only weak PARP-1 inhibiting activity, whereas the caffeine metabolites 1,7-dimethylxanthine, 3-methylxanthine and 1-methylxanthine, as well as theobromine and theophylline showed significant PARP-1 inhibiting activity. Further evaluation of these compounds in H 2O 2-treated A549 lung epithelial and RF24 vascular endothelial cells revealed that the decrease in NAD +-levels as well as the formation of the poly(ADP-ribose)polymer was significantly prevented by the major caffeine metabolite 1,7-dimethylxanthine. Furthermore, H 2O 2-induced necrosis could be prevented by a high dose of 1,7-dimethylxanthine. Finally, antioxidant effects of the methylxanthines could be ruled out with ESR and measurement of the TEAC. Concluding, caffeine metabolites are inhibitors of PARP-1 and the major caffeine metabolite 1,7-dimethylxanthine has significant PARP-1 inhibiting activity in cultured epithelial and endothelial cells at physiological concentrations. This inhibition could have important implications for nutritional treatment of acute and chronic inflammatory pathologies, like prevention of ischemia-reperfusion injury or vascular complications in diabetes.

ANTIRADICAL EFFICIENCY OF TEA COMPONENTS

ABSTRACTTea contains antioxidant compounds, such as (‐)‐epicatechin (EC), (‐)‐epigallocatechin (EGC), (‐)‐epicatechin gallate (ECG), (‐)‐epigallocatechin gallate (EGCG), gallic acid and caffeine. The antioxidant activity of these compounds as well as 1,3,7‐trimethyluric acid (the major metabolite of caffeine) was evaluated using the 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH). The antiradical ability according to the EC50 was EGCG > ECG > EGC > gallic acid > EC > BHT > 1,3,7‐trimethyluric acid > caffeine. The time for these compounds to reach the steady state was considered, and the concept of antioxidant efficiency (AE50) involving the EC50 and the time to reach the steady state was used to evaluate the antiradical efficacy. The order of antiradical efficiency according to AE50 was ECG gallic acid > EGCG > EC > EGC > BHT > 1,3,7‐trimethyluric acid > caffeine.

Ｅｈｒｌｉｃｈ腫よう細胞におけるａｄｒｉａｍｙｃｉｎ ｅｆｆｌｕｘに対するｃａｆｆｅｉｎｅの影響

We previously reported that caffeine inhibited adriamycin efflux in Ehrlich ascites carcinoma cells and it would be of value as biochemical modulator of adriamycin. In this study, the transport of adriamycin was investigated, in vitro, as a part of a study to clarify the mechanism of adriamycin efflux inhibition by caffeine, using metabolites of caffeine. The mechanism of the effect of caffeine on adriamycin antitumor activity, in vivo, was also studied.1, 3, 7-Trimethyluric acid did not inhibit adriamycin efflux on tumor cells in vitro, and did not enhance the antitumor activity of adriamycin in vivo. Furthermore, 1, 7-dimethylxanthine, which is the major caffeine metabolite in human, promoted adriamycin efflux. However, theobromine would be of value as biochemical modulator of adriamycin from in vitro and in vivo study. Moreover, when the temperature of incubation was 20°C, the inhibition of adriamycin efflux by caffeine did not observed, It suggest that some enzymes system in the cell will be related to adriamycin efflux. However, since ouabain had no effect on the adriamycin efflux, Na+, K+-ATPase do not concern with adriamycin efflux. And in medium without glucose, there were no effects of caffeine. It suggested that energy which supply from glucose is needed to adriamycin transport. These results suggest that the effect of caffeine, as biochemical modulator, is not due to metabolite of caffeine. And the transport of adriamycin and effects of caffeine is considered to be related to some enzymes and to some energy consumptions in the cell.

Effect of Chronic Caffeine Administration on Theophylline Concentrations Required to Produce Seizures in Rats

Caffeine as well as the antiasthmatic drug theophylline can cause seizures when administered to humans or animals in excessive doses. Studies on rats have shown rapid development of functional tolerance to caffeine-induced seizures whereas repeated pretreatment with theophylline had no significant effect on the theophylline concentrations required to produce seizures. The purpose of this investigation was to determine whether chronic exposure to caffeine can affect susceptibility to the convulsant effect of theophylline. Rats received caffeine, 40 mg/kg, or solvent twice a day for 7 days as an intravenous injection. On the eighth day, theophylline was infused intravenously until the onset of maximal seizures. At this pharmacologic end point, rats pretreated with caffeine had significantly higher theophylline concentrations in the brain and cerebrospinal fluid than did control (solvent-pretreated) animals. Although the concentration differences were relatively small (approximately 11%), they demonstrate in principle the development of caffeine-induced tolerance to the neurotoxic effect of theophylline. Additional experiments showed that the caffeine effect on theophylline neurotoxicity is not acutely mediated by paraxanthine, a major metabolite of caffeine.

Separation and determination of theophylline from paraxanthine in human serum by reversed-phase high-performance liquid chromatography

A sensitive and highly specific method for the determination of theophylline in serum by high-performance liquid chromatography (HPLC) has been developed. Theophylline was completely separated from paraxanthine, a major metabolite of caffeine which has been known to interfere with most isocratic reversed-phase HPLC methods, with a mixture of acetonitrile/tetrahydrofuran/acetate buffer (10 mM, pH 5.0; 5:1:94, v/v %) as the mobile phase using a C 18 bonded reversed-phase column. Neither the other xanthine and uric acid derivatives nor numerous drugs tested were found to interfere. The proposed method was applied to therapeutic monitoring utilizing its excellent precision, reproducibility and high specificity for theophylline.

Methylxanthines: Toxicity to humans. 3. Theobromine, paraxanthine and the combined effects of methylxanthines

This review provides a brief overview of known information on the human toxicity of theobromine and paraxanthine. Theobromine has some pharmacological effects, although these activities are considerably weaker than those of theophylline and/or caffeine, described in parts 1 and 2 of this series (Stavric, Fd Chem. Toxic. 1988, 26, 541 & 645). Paraxanthine, which is not found in plants or foods, is the major metabolite of caffeine in humans, in whom its toxicological potency appears to be very low. This paper gives a brief retrospective view of possible toxicological effects when methylxanthines are taken simultaneously or are present in combination as a result of metabolic transformation. Critical review of toxic manifestations due to exposure to relatively large doses of caffeine and theophylline indicates that such combined exposure may potentiate the toxic effects of either drug.

Quantitative assessment of caffeine partial clearances in man.

Five subjects who participated in an earlier study (Lelo et al., 1986b) of the comparative pharmacokinetics of caffeine (CA) and its primary monodemethylated metabolites paraxanthine (PX), theobromine (TB) and theophylline (TP) were administered CA to steady-state. Using areas under the plasma concentration-time curves for each of the dimethylxanthines derived from CA in the steady-state study and individual plasma clearances of PX, TB and TP determined in the previous study, the fractional conversion of CA to PX, TB and TP and the individual partial clearances of CA have been defined. The mean (+/- s.d.) fractional conversion of CA to PX, TB and TP was 79.6 +/- 21.0%, 10.8 +/- 2.4% and 3.7 +/- 1.3%, respectively. When only demethylation pathways are considered PX, TB and TP accounted for 83.9 +/- 5.4%, 12.1 +/- 4.1% and 4.0 +/- 1.4%, respectively of the CA demethylations. The mean partial clearance of CA to PX was approximately 8-fold and 23-fold greater than those to TB and TP respectively. These data confirm earlier reports that PX is the major metabolite of CA in humans but suggest that PX formation is quantitatively more important than previously believed.

Placental transfer of the major caffeine metabolite in the rat using 6-amino-5[ N-formylmethylamino]1,3[ Me- 14C]-dimethyluracil administered orally or intravenously to the pregnant rat

6-Amino-5[ N-formylmethylamino]1,3[ Me- 14C]dimethyluracil (1,3,7-DAU), the most important caffeine metabolite in the rat and a minor one in man was synthesized and administered p.o. or i.v. to pregnant rats. This study demonstrates the distribution of this metabolite in the animal and its transfer to the embryos and the fetus. The fetus was shown to be protected by a placental barrier which leads to a lower fetal tissue exposure 1 h after the administration, the equilibrium between fetus and pregnant rat being reached 4–5 h later. Future studies testing the fetotoxicity of this metabolite compared with caffeine must take into consideration that only about half of the oral dose is absorbed. In addition, similar fetal tissue exposure must be obtained when this metabolite is given orally or is produced from caffeine.

Caffeine-metabolizing capability of primary rat hepatocytes, Chinese hamster V79 cells and normal human fibroblasts in culture

Caffeine is metabolized by primary rat hepatocytes in culture to metabolites similar to those found in rats in vivo, including 6-amino-5-[ N-formylmethylamino]-1,3-dimethyluracil, one of the major caffeine metabolites produced in the rat. At a substrate-limiting level of caffeine, the rat hepatocytes metabolized 25–30% of the total added caffeine in 24 h. About 99% of the metabolites was found in the extracellular medium and rinse fractions, suggesting that on a volume basis, caffeine metabolites are in equilibrium inside and outside the cells. No detectable caffeine metabolites were produced in preparations of either Chinese hamster V79 cells, or normal human fibroblasts at caffeine concentrations from 5 μM to 5 mM. These results suggest that the varied biological effects induced by caffeine in Chinese hamster cells cannot be attributed to caffeine metabolites and further that the differential responses of Chinese hamster cells and normal human fibroblasts to caffeine are not due to qualitative or quantitative differences in caffeine metabolism in the two cell lines.

In vitro and in vivo cytotoxicity of 6 amino-5-formylmethylamino-1,3-dimethyl uracil, a uracilic metabolite of caffeine

The in vitro cytotoxicity of 6 amino-5 formylmethylamino-1,3 dimethyluracil (ADMU) a major metabolite of caffeine in rats was studied by cell counts or [ 3H]thymidine incorporation in the murine Lewis lung carcinoma (3LL) and L929 fibroblast cells and in the human E cell line derived from an ovarian carcinoma. Unlike 5-fluorouracil (5FU) which was markedly cytotoxic, ADMU concentrations up to 60 μg/ml were devoid appreciable cytocidal action. Similarly, 1–10 μg 5FU markedly inhibited the blastogenic response of rat lymphocytes to PHA, whereas lymphoproliferation was not affected at ADMU concentrations up to 100 μg/ml. In vivo administration of ADMU (40 mg/kg, twice a day on day 1 to 3) to L1210 leukaemia-bearing mice caused a transient short-lasting reduction of tumour cell numbers only on day 6 after leukaemia inoculation.